DESCRIPCIÓN DE LA PROBLEMÁTICA

A growing body of preclinical and clinical evidence indicates that integrin αVβ3 is a promising target for anti-angiogenic therapy. Several integrin αVβ3-targeted therapeutic approaches such as the direct signaling inhibition using antibodies, peptides, peptidomimetics, and other antagonists, as well as indirect tumor-targeted drug delivery systems have been summarized. Since integrin αVβ3 is not the only molecule involved in tumor angiogenesis, the combined inhibition of integrin αVβ3 with other angiogenic molecules such as VEGF, FGF, EGF, and other integrins is expected to generate significantly greater anti-angiogenic and anti- tumor benefits over monospecific αVβ3 antagonists. Additionally, the inhibition of integrin αVβ3 combined with other therapeutic modalities, such as chemotherapy and radiotherapy, should be much more effective than using integrin αVβ3 alone. Importantly, this study provides an example of proof-of-concept targeting a single agent to target tumor microenvironment by harnessing both anti-angiogenic and anti-fibrotic activity. The developed agent, ProAgio, is not toxic to established vasculature, inactive fibroblasts, inactive HSCs, resting pancreatic stellate cells and fenestrated sinusoidal endothelial cells. But all the above-mentioned cell types once

126 activated promote the pathological progression of fibrosis and related diseases, cancer being the common one. ProAgio is a potential candidate for clinical applications in various diseases as demonstrated by various in vitro and in vivo experiments. However, the specific synergistic interactions among different modalities should be investigated and clearly understood for the personalized treatment.

Targeting the microenvironment in Hepatocellular carcinoma - Tumor microenvironment has a crucial role in HCC development and progression. Modulating the dynamic crosstalk between stroma and hepatocytes as a treatment for HCC holds a very strong rationale, especially in HCC prevention. Liver fibrosis reversal using antifibrotic therapies has been shown in patients with chronic liver diseases. Also, targeting of various immune cells and specific molecules that are present in the pro-tumorigenic microenvironment in the liver is also feasible. Despite these options, enhanced understanding of the relative contribution of different molecules and cells contributing to HCC pathogenesis, and interpretation of the hierarchy of pathological mechanisms occurring in liver, are essential for planning preventive therapies or therapeutics. This will require an improved comprehension of the various complex interactions that occur in chronically inflamed human livers, along with careful testing of their functional significance in animal models that could translate to different etiologies of hepatic inflammation (metabolic, viral etc.) in humans. The ideal preventive treatment in HCC should change a pro-tumorigenic microenvironment into an anti-tumorigenic one. It has been clearly demonstrated that hepatic fibrosis and cirrhosis play a pivotal role in causing HCC. Several mechanisms through which activated hepatic stellate cells lead to HCC progression include increased tissue stiffness, angiogenesis, and secretion of cytokines, pro-tumorigenic extracellular matrix components and immunosuppression. However, these mechanisms and their contribution in HCC in concert to humans remains to be elucidated. In summary, chronic liver inflammation generates a pro- tumorigenic microenvironment, infiltrated by stellate cells, immune cells, together producing a pathological milieu, which is comprised of multiple extracellular matrix proteins, collagens, cytokines and growth factors that can form a pro-tumorigenic stroma. Hence, targeting the microenvironment could be a promising therapeutic approach.

127

Targeting the microenvironment in Pancreatic ductal adenocarcinoma – Pancreatic cancers have an abundant and dense collagenous stroma, also known as desmoplasia, that results in hypoxia in cancer cells. This stroma is composed of extracellular matrix proteins such as collagens, laminin and fibronectin as well as non-collagenous proteins such as glycoproteins, glycosaminoglycans and proteoglycans. The factors in the stroma that probably mediate the interaction of cancer cells with the ECM include growth factors, periostin, osteopontin and serine protein acidic and rich in cysteine. Similar to HCC, pancreatic cancer stellate cells (PSCs) form the pathological microenvironment in pancreatic cancer. PSCs produce collagenous matrix, aid in infiltration of immune cells, endothelial cells and neuronal cells. However, the co-relation between extent of stromal expansion and the clinical outcome is debatable. The association between the numbers of activated pancreatic stellate cells and poor clinical outcome was reported in several studies individually, pancreatic cancer patients showed that in early-stage (T1–T2) pancreatic tumors, moderate or strong α-sma expression was related with poorer clinical outcomes than tumors with low levels of α-sma expression, as evaluated by overall and progression-free survival.

The interaction between cancer cells and stromal stellate cells has been demonstrated by both in vitro and in vivo studies. Stellate cells can inhibit apoptosis of cancer cells, increase their survival, and aid cancer stem cell niche formation that possibly has a role in recurrence of the disease and chemo-resistance. In addition, PSCs can metastasize from the primary tumor to distant sites, where they might facilitate the seeding and progression of metastatic cancer cells. It is important clinically because factors that ward off immune system such programmed cell death protein ligand 1 (PD-L1), expressed on cancer cells form the basis of novel immunotherapeutic strategy that is currently under investigation in pancreatic cancer.

Targeting the microenvironment in lung metastasis – Lung metastasis is the most commonly observed metastatic site for various cancers including breast liver, pancreas and prostate. Homing of circulating cancer cells is enhanced in lung due to the microenvironment which has a rich supply of nutrients due to dispersed capillaries, high oxygen and moreover the fibroblasts in lung which secrete collagen and form a premetastatic niche which aid to colonize the circulating tumor cells. We believe that this activation of lung tissue provides an ideal niche for colonizing

128 the metastatic cells. It is not yet clear whether cancer cells go to the metastatic site and activate or they activate and colonize. This study will establish the story of breast cancer metastasizing to lungs with and with our pre-activation. This pre-metastatic niche mainly comprises of ECM and fibroblasts. In orthotopic breast cancer 4T1 metastatic breast cancer model we studied we have observed remarkable decrease in lung metastasis which we mainly attribute to the decreased activated lung fibroblasts. It has always been challenging to regulate the tumor growth. Starting with a chemotherapy specifically killing cancer cells, then with additional anti angiogenic therapy and still the cancer progression has lot more factors than anticipated. To our recent understanding of tumor microenvironment, it came to light that not only cancer cells and endothelial cells but also fibroblasts, pericytes and stem cells also known to play a prominent role in cancer progression. It has always been challenging to target resident fibroblasts or fibroblasts formed from other sources such as pericytes, stem cells or epithelial cell derived. TGF-β and TGF-βR even though are responsible for its major function couldn't become optimistic targets. We believe that integrin v3 which is also highly expressed in these cells could be most viable option as v3 is expressed on endothelial cells, fibroblasts and pericytes. Making a potential drug that can effectively target this receptor can effectively have a great impact on tumor microenvironment enhancing both chemotherapy and immunotherapy.

To summarize, a novel anti-angiogenic and anti-fibrotic agent has been successfully developed by rational protein design. Cancer therapeutics are moving away from cytotoxic to specifically targeted molecules having minimum side effects and ProAgio will serve as a perfect therapeutic agent as it will address angiogenesis and its related diseases such as age related macular degeneration, cirrhosis and fibrosis (liver and pancreatic fibrosis). It will also be effective in various cancers where angiogenesis and fibrosis are either a cause or a consequence, especially in life threatening cancers such as pancreatic cancer, HCC, lung cancer and various other organ related cancers. In the aforementioned cases, tumor microenvironment plays a prominent role in tumor growth and cancer progression and there is no cure available yet. ProAgio has also exhibited significant therapeutic efficacy in regulating metastatic progression in various models discussed in this thesis.

129 ProAgio has a wide range of applications in pathological conditions beyond any known therapeutic agent. Therefore, a careful and thorough analysis has to be performed before putting it into the clinic. Historically, not all animal models faithfully translate to the human state, (exemplified by the disparate results of TNFα inhibition in rodents as compared to human alcoholic liver disease, thalidomide for morning sickness, endostatin and cilengitide for tumor xenografts). Clearly, human investigations with all proper safety measures need to be pursued.

“With great power comes great responsibility” François-Jean Baudouin May 8, 1793.

Thank you Ravi Chakra Turaga

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